PROJECT SUMMARY Mitochondrial dysfunction is an underlying cause of cardiomyocyte death and therefore plays a critical role in the development of many cardiac pathologies. This mitochondrial dysfunction is often mediated by the opening of the mitochondrial permeability transition (MPT) pore, which causes a rapid increase in inner mitochondrial membrane permeability. This in turn leads to ATP depletion, reactive oxygen species production, mitochondrial swelling and rupture. Consequently, our long-range goal is to identify the proteins that make up the MPT pore and understand the molecular mechanisms by which this complex is regulated. The MPT pore was originally proposed to consist of the voltage-dependent anion channel (VDAC) in the outer membrane, the adenine nucleotide translocase (ANT) in the inner membrane, plus a regulatory protein cyclophilin-D (CypD) in the matrix. However, genetic studies have revealed that VDAC and ANT are dispensable for MPT. Thus CypD still remains as the only bona fide member of the MPT pore complex. Importantly, the key factors that directly regulate CypD and its pro-MPT function remain to be elucidated. In particular, CypD-binding proteins that act to inhibit CypD's function have yet to be identified and the additional role that CypD phosphorylation plays in regulating MPT and cell death has not been comprehensively addressed. We have identified the mitochondrial matrix protein C1qbp as a novel CypD-binding protein and our strong preliminary data indicate that it can inhibit MPT and cell death. Conversely, we have found that the pro-death kinase GSK3 can sensitize cells to MPT and death, and that these effects are associated with CypD phosphorylation. Consequently, our central hypothesis is that C1qbp inhibits, whereas GSK3 promotes, MPT and cell death through the direct regulation of CypD. The objective of the present application is to utilize genetic, biochemical, physiological, and pharmacological techniques to systematically evaluate the roles of a CypD inhibitor (C1qbp) and a CypD activator (GSK3) in MPT, cardiac cell death, and the progression of myocardial disease. In Aim 1 we will determine the functional involvement of C1qbp in the MPT response and cardiac myocyte death. In Aim 2 we will evaluate the effects of GSK3-dependent CypD phosphorylation on MPT and cardiac cell death. The rationale for the proposed research is that once key mitochondrial proteins that regulate MPT and mitochondrial function are identified, they can be targeted as a means of treating a whole array of human cardiac diseases.